Methods of therapy with thrombin derived peptides

ABSTRACT

The present invention relates to a method for promoting cardiac tissue repair comprising administering to the cardiac tissue a therapeutically effective amount of an angiogenic thrombin derivative peptide and/or inhibiting or reducing vascular occlusion or restenosis. The invention also relates to methods of stimulating revascularization. In yet another embodiment, the invention relates to the use of thrombin derivative peptides in the manufacture of a medicament for the methods described herein.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/957,427, filed Sep. 30, 2004, which is a continuation of U.S.application Ser. No. 10/050,611, filed Jan. 16, 2002, which is acontinuation-in-part of U.S. application Ser. No. 09/904,090, filed Jul.12, 2001, which claims the benefit of U.S. Provisional Application No.60/217,583, filed Jul. 12, 2000. The entire teachings of the aboveapplications are incorporated herein by reference.

GOVERNMENT SUPPORT

The invention was supported, in whole or in part, by a grant R43 HL64508from National Institutes of Health. The Government has certain rights inthe invention.

BACKGROUND OF THE INVENTION

Human alpha-thrombin appears to have growth-promoting activity for awide variety of cells from various tissues. For example, alpha-thrombinhas been shown to initiate proliferation of fibroblastic cells inculture without addition of serum or other purified growth factors, tosynergize with epidermal growth factor in certain hamster fibroblastsand human endothelial cells, to initiate cell division or DNA synthesisin mammalian lens epithelial and spleen cells and actuate monocytes andneutrophils. Yet, the use of thrombin as a growth factor and itspotential importance to wound healing has not been widely acclaimed. Inpart, this may be due to the complexity of thrombin's involvement withcoagulation, platelet activation, and initiation of cell proliferationas well as to the complex regulation of thrombin and thrombin-likemolecules by serum protease inhibitors and by cell-released proteasenexins. This complexity and high degree of physiologic regulation,however, supports the potential importance of this initiation pathway inwound healing.

Thrombin may also play a role in both normal revascularization andmigration of cells from the blood to the site of injury and the abnormalmetastasis and angiogenesis associated with tumors. The ability ofthrombin to increase endothelial cell proliferation and alter thebarrier function of blood vessels may contribute to angiogenesis andinflammation at sites of tissue injury.

Thrombin derivative peptides have been described by the presentinventors for the agonizing and antagonizing thrombin and/or thrombinreceptor activity, such as in the treatment of wounds. U.S. Pat. No.5,500,412 or 5,352,664, the contents of which are incorporated herein byreference in their entirety. However, the patent does not teach thenovel use of the thrombin derivative peptides for the treatment ofdamaged cardiac tissue, for revascularization, or for inhibition ofvascular occlusion and restenosis.

SUMMARY OF THE INVENTION

The invention relates to methods for promoting cardiac tissue ormyocardium repair, promoting vascularization or inhibiting vascularocclusion or restenosis. The method comprises administering to thecardiac tissue or blood vessels a therapeutically effective amount of anangiogenic thrombin derivative peptide. In a preferred embodiment, thepeptide is a peptide described in U.S. Pat. No. 5,500,412 or 5,352,664,the contents of which are incorporated herein by reference in theirentirety. For example, the peptide can preferably comprises a thrombinreceptor binding domain having the sequence Arg-Gly-Asp-Ala (SEQ ID NO:1); and a serine esterase conserved sequence. Preferred serine esteraseconserved sequences compriseAsp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val (SEQ ID NO: 2). In yet amore preferred embodiment, the thrombin derivative peptide comprises theamino acid sequence:Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val(SEQ ID NO: 3), such as a peptide which consists of the amino acidsequenceAla-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val(SEQ ID NO: 3). The peptide having the sequence of SEQ ID NO. 3 is alsoreferred to herein as “TP508”).

Alternatively, the thrombin derivative peptide is a physiologicallyfunctional equivalent of the thrombin derivative peptide comprising theamino acid sequence of SEQ ID NO: 3. In a particular embodiment, thethrombin derivative peptide comprises the modified amino acid sequence:Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val-NH₂(SEQ ID NO: 4). In a particular embodiment, the thrombin derivativepeptide consists of the modified amino acid sequence:Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val-NH₂(SEQ ID NO: 4).

The peptide can preferably be administered during or following cardiacsurgery, for example by direct or catheter-mediated injection intodamaged or ischemic cardiac tissue as a soluble peptide or in asustained release formulation.

The invention also relates to a method of stimulating revascularizationor vascular endothelial cell proliferation comprising administering tocardiac tissue a therapeutically effective amount of an angiogenicthrombin derivative peptide, as described herein.

The invention also relates to a method of preventing vascular occlusionor restenosis comprising administering a therapeutically effectiveamount of the angiogenic thrombin receptor binding peptide to bloodvessels, for example, by systemic injection, by delivering the peptidesto sites of vascular injury by catheter, or by attachment of the peptideto stents.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing that increasing concentrations of TP508(peptide having the amino acid sequence of SEQ ID NO: 3) stimulates theproliferation of human microvascular endothelial cells in vitro. Thegraph shows the cell count 48 hours after being administered variousconcentrations of TP508 (indicated in μg/ml).

FIG. 2 is a graph showing that increasing concentrations of TP508stimulates the migration of microvascular endothelial cells on plastic.The graph shows the distance migrated by the cells after beingadministered various concentrations of TP508 (indicated in μg/ml).

FIG. 3 is a graph showing changes in cardiac function in TP508 treatedand control pigs in porcine model of cardiac ischemia.

DETAILED DESCRIPTION OF THE INVENTION

Cardiovascular diseases are generally characterized by an impairedsupply of blood to the heart or other target organs. Myocardialinfarction (MI) result from narrowed or blocked coronary arteries in theheart which starves the heart of needed nutrients and oxygen. When thesupply of blood to the heart is compromised, cells respond by generatingcompounds that induce the growth of new blood vessels so as to increasethe supply of blood to the heart. These new blood vessels are calledcollateral blood vessels. The process by which new blood vessels areinduced to grow out of the existing vasculature is termed angiogenesis,and the substances that are produced by cells to induce angiogenesis arethe angiogenic factors.

When heart muscle is deprived of oxygen and nutrients due to vascularocclusion, the heart muscle tissue becomes ischemic and loses itsability to contract and function. This loss of function may be restoredby natural signals from the ischemic heart muscle that induce angiogenicrevascularization through development of collateral vessels that bypassthe occlusion. This revascularization or angiogenesis involves thestimulation of endothelial cell proliferation and migration and buddingoff of new blood vessels. In many cases, however, the natural signalsare not sufficient to cause collateral vessel growth and the ischemictissue can become fibrotic or necrotic. If this process is not reversedby procedures to open the occluded vessels or further induction ofcollateral blood vessels, the heart may become totally disfunctional andrequire transplantation.

The peptides described herein can be employed to induce angiogenicproliferation and migration of endothelial cells resulting in formationof new capillaries and collateral vessels to help restore function todamaged or ischemic heart tissue. These peptides may preferably bedirectly injected into or applied to heart tissue during open chestprocedures for bypass surgery or insertion of ventricular assist devicesor delivered by catheter injection into the heart as a soluble peptideor in a sustained release formulation.

Endothelial cell proliferation, such as that which occurs inangiogenesis, is also useful in preventing or inhibiting restenosisfollowing balloon angioplasty. The balloon angioplasty procedure ofteninjures the endothelial cells lining the inner walls of blood vesselsand disrupts the integrity of the vessel wall. Smooth muscle cells andinflammatory cells often infiltrate into the injured blood vesselscausing a secondary obstruction in a process known as restenosis.Stimulation of the proliferation and migration of the endothelial cellslocated at the periphery of the balloon-induced damaged area in order tocover the luminal surface of the vessel with a new monolayer ofendothelial cells would potentially restore the original structure ofthe blood vessel.

Preferably, endothelialization comprises re-endothelialization afterangioplasty, to reduce, inhibit or prevent restenosis. Those of skill inthe art will recognize that patients treated according to the methods ofthe present invention may be treated with or without a stent.

An inflatable balloon catheter with peptide coating the balloon or acatheter that directly injects the peptide into the wall of the vesselmay also be employed to deliver the substance to a targeted artery.

Balloon angioplasty is a common treatment of ischemic heart diseasewhich involves the inflation of a balloon in a clogged blood vessel inorder to open the blocked blood vessel. Unfortunately, this method oftreatment results in injury to the endothelial cells lining the innerwalls of blood vessels often leading to restenosis. The peptidesdescribed herein can be employed to induce proliferation and migrationof the endothelial cells located at the periphery of the balloon induceddamaged area in order to cover the luminal surface of the vessel with anew monolayer of endothelial cells, hoping to restore the originalstructure of the blood vessel. Coronary angioplasty is frequentlyaccompanied by deployment of an intravascular stent to help maintainvessel function and avoid restenosis. Stents have been coated withheparin to prevent thrombosis until the new channel formed by the stentcan endothelialize. The peptides described herein can be applieddirectly to the stent, using methods known to those of skill in the art.The peptides can be locally applied or systemically administered toenhance endothelialization of the vessel or vessel wall and/or tomodulate other processes to inhibit or reduce thrombosis and restenosis.

The present invention preferably employs synthetic or naturally derivedpolypeptide agonists of thrombin receptor mediated events. Both of theseclasses of agents possess a thrombin receptor binding domain whichincludes a segment of the polypeptide that is capable of selectivelybinding to the high-affinity thrombin receptor. This segment of thepolypeptide includes a sequence of amino acids homologous to atripeptide cell binding domain of fibronectin.

In addition to the thrombin receptor binding domain, the stimulatory(agonistic) polypeptides possess a sequence of amino acids havingsequences derived from the N-terminal amino acids of a dodecapeptidepreviously shown to be highly conserved among serine proteases. However,the inhibitory polypeptides do not include these serineesterase-conserved sequences.

For example, the invention provides a number of polypeptides useful inpromoting cardiac tissue repair. For such applications, the inventionprovides a polypeptide derivative of thrombin (or a functionalequivalent of such a derivative) which has a thrombin receptor bindingdomain as well as a domain with a serine esterase conserved sequence ofat least 12 amino acids. The invention also provides a polypeptidecompound of at least 23 L-amino acids which has both a thrombin receptorbinding domain and a domain with a serine esterase conserved amino acidsequence.

In one embodiment, the invention provides for several polypeptidescontaining specific amino acid sequences, such as a polypeptide compoundin which the thrombin receptor binding domain includes the L-amino acidsequence Arg-Gly-Asp-Ala (SEQ ID NO: 1) together with the serineesterase conserved amino acid sequence,Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val (SEQ ID NO: 2). In apreferred embodiment, the polypeptide compound includes the L-amino acidsequenceAla-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val(SEQ ID NO: 3). The polypeptide compound can be modified by amidation ofthe carboxy terminus. For example, SEQ ID NO: 3 can be amidated asfollows:Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val-NH2(SEQ ID NO: 4).

The invention also provides for a pharmaceutical composition forpromoting tissue repair which includes a therapeutically effectiveconcentration of any of the compounds described above combined with apharmaceutically acceptable excipient. Typically, such compositionsinclude, for example, sufficient concentrations of the polypeptides toeffect a stimulatory action on the thrombin receptor as demonstratedherein. Thus, such compositions should typically include sufficientconcentrations to obtain levels of the polypeptides at the target sitewhich are shown in vitro to stimulate the receptor. When endogenouslevels of a secondary signal are believed to be inadequate, compositionsmay be employed which further include the addition of a therapeuticallyeffective concentration of VEGF, alpha-thrombin, gamma-thrombin or othergrowth factors. Such combinations may exert an additive or synergisticeffect. In certain cases, if tissue damage is so extensive that cellscapable of responding to the polypeptides are not present in sufficientquantities, it is expected that responsive cells could be co-injected toprovide a therapeutically effective combination.

Suitable carriers also provide for release of the active ingredient andpreferably for a slow, sustained release over time at the target site. Anumber of synthetic biodegradable polymers can serve as carriers withsustained release characteristics. Examples of these polymers includepoly α-hydroxy esters such as polylactic acid/polyglycolic acidhomopolymers and copolymers, polyphosphazenes (PPHOS), polyanhydridesand poly(propylene fumarates).

Polylactic acid/polyglycolic acid (PLGA) homo and copolymers are wellknown in the art as sustained release vehicles. The rate of release canbe adjusted by the skilled artisan by variation of polylactic acid topolyglycolic acid ratio and the molecular weight of the polymer (seeAnderson, et al., Adv. Drug Deliv. Rev. 28:5 (1997), the entireteachings of which are incorporated herein by reference). Theincorporation of poly(ethylene glycol) into the polymer as a blend toform microparticle carriers allows further attenuation of the releaseprofile of the active ingredient (see Cleek et al., J. Control Release48:259 (1997), the entire teachings of which are incorporated herein byreference). PGLA microparticles are often mixed with pluronic gels orcollagen to prevent aggregation and to make the microparticles suitablefor direct injection.

PPHOS polymers contain alternating nitrogen and phosphorous with nocarbon in the polymer backbone, as shown below in Structural Formula(I):

The properties of the polymer can be adjusted by suitable variation ofside groups R and R′ that are bonded to the polymer backbone. Forexample, the degradation of and drug release by PPHOS can be controlledby varying the amount of hydrolytically unstable side groups. Withgreater incorporation of either imidazolyl or ethylglycinato substitutedPPHOS, for example, an increase in degradation rate is observed (seeLaurencin et al., J Biomed Mater. Res. 27:963 (1993), the entireteachings of which are incorporated herein by reference), therebyincreasing the rate of drug release.

Polyanhydrides, shown in Structural Formula (II), have well defineddegradation and release characteristics that can be controlled byincluding varying amounts of hydrophobic or hydrophilic monomers such assebacic acid and 1,3-bis(p-carboxyphenoxy)propane (see Leong et al., J.Biomed. Mater. Res. 19:941 (1985), the entire teachings of which areincorporated herein by reference).

Poly(propylene fumarates) (PPF) are highly desirable biocompatibleimplantable carriers because they are an injectable, in situpolymerizable, biodegradable material. “Injectable” means that thematerial can be injected by syringe through a standard needle used forinjecting pastes and gels. PPF, combined with a vinyl monomer (N-vinylpyrrolidinone) and an initiator (benzoyl peroxide), forms an injectablesolution that can be polymerized in situ (see Suggs et al.,Macromolecules 30:4318 (1997), Peter et al., J. Biomater. Sci. Poly,.Ed. 10:363 (1999) and Yaszemski et al., Tissue Eng 1:41 (1995), theentire teachings of which are incorporated herein by reference).

As used herein, a therapeutically effective concentration is defined asa concentration of the particular agent which provides a satisfactoryincrease in the rate of repair or angiogenesis or which provides asatisfactory reduction or inhibition of restenosis or vascularocclusion. Again, such concentrations are believed to correspond tolevels sufficient to elicit a stimulation of the high-affinity thrombinreceptor in vitro. However, it is believed that the compositions willprove most effective when the stimulatory (agonistic) polypeptides arepresent at a concentration of from 0.1 μM to 10 μM.

For purposes of the present invention, a thrombin derivative is definedas any molecule with an amino acid sequence derived at least in partfrom that of thrombin, whether synthesized in vivo or in vitro.Accordingly, a thrombin derivative, as referred to herein, designates apolypeptide molecule which comprises fewer amino acids than thrombin.

A physiologically functional equivalent of a thrombin derivativeencompasses molecules which differ from thrombin derivatives inparticulars which do not affect the function of the thrombin receptorbinding domain or the serine esterase conserved amino acid sequence.Such particulars may include, but are not limited to, conservative aminoacid substitutions and modifications, for example, amidation of thecarboxyl terminus, acetylation of the amino terminus, conjugation of thepolypeptide to a physiologically inert carrier molecule, or sequencealterations in accordance with the serine esterase conserved sequences.

A thrombin receptor binding domain is defined as a polypeptide sequencewhich directly binds to the thrombin receptor and/or competitivelyinhibits binding between high-affinity thrombin receptors andalpha-thrombin.

A domain having a serine esterase conserved sequence comprises apolypeptide sequence containing at least 4-12 of the N-terminal aminoacids of the dodecapeptide previously shown to be highly conserved amongserine proteases (Asp-X₁-Cys-X₂-Gly-Asp-Ser-Gly-Gly-Pro-X₃-Val-SEQ IDNO: 5); wherein X₁ is either Ala or Ser; X₂ is either Glu or Gln; and X₃is either Phe, Met, Leu, His, or Val).

A stimulatory polypeptide is defined as a polypeptide derivative ofthrombin, or a physiologically functional equivalent thereof, having theability to both bind to and stimulate the thrombin receptor. Therefore,the stimulatory peptides will include both a thrombin receptor bindingdomain and a domain with a serine esterase conserved amino acidsequence.

The invention is illustrated by the following examples, which are notintended to be limiting in any way.

EXEMPLIFICATION Example 1 TP508 Stimulates the Proliferation andMigration of Human Endothelial Cells In Vitro

To determine if TP508 could directly induce proliferation of endothelialcells, human microvascular endothelial cells were purchased fromClonetics, plated on tissue culture grade plastic in 24 well culturedishes and serum starved for 24 hours. Cells were stimulated in mediumwith or without TP508 for 48 hours, at which time proliferation wasassessed using a direct cell count. As shown in FIG. 1, TP508 stimulatedproliferation of microvascular endothelial cells by 30 to 50% over thosetreated in medium alone (1.0 μg/ml TP508). This effect appeared to bespecific since the growth of smooth muscle cells isolated from rat aortawas not affected by TP508.

TP508 effects on migration of human endothelial cells was assessed usingan in vitro monolayer wound assay in which endothelial cells were platedin 35 mm culture dishes and allowed to grow to near confluency for threedays, at which time the monolayer was “wounded” by scraping across thecenter of the dish with a rubber policeman to remove a band of cells.Photographs were taken at this point, and the cells were then treatedwith fresh medium alone or medium containing various concentrations ofTP508 and allowed to grow for an additional 48 hours. The cells werere-photographed, and the distance that the endothelial cells migratedinto the wounded area was measured. As shown in FIG. 2, TP508 stimulatedmigration of endothelial cells, even when the cells were cultured onplastic alone.

These studies demonstrated that TP508 has direct angiogenic effects onhuman endothelial cells causing increased proliferation and migration invitro. Additional studies indicate that exposure of endothelial cells toTP508 has a protective effect to prevent death of cells caused byoxidative exposure. This protective effect may also contribute toprocesses of re-endothelialization and angiogenesis.

Example 2 TP508 Stimulates Angiogenesis In Vitro in a ChorioalloantoicMembrane Model

Studies with full dermal surgical incisions and open excisional woundsin the backs of rats showed that a single topical application of TP508stimulates revascularization and the patency of blood vessels traversinga surgical incision. Two surgical incisional wounds were made on theback of a rat. One wound was treated with a single application of TP508(0.1 μg); the other was untreated. Blood vessels were attracted to thetreated wound rather than the control.

Addition of TP508 to agar disks placed on the chorioalloantoic membraneof chicken embryos resulted in an angiogenic outgrowth of blood vessels.Blood vessels were stimulated to grow into agar disks containing TP508.There was also an increase in collateral vessel outgrowth in vesselsdistal to the plug similar to that observed with other angiogenicfactors.

Example 3 TP508 Showed Efficacy in Treating Myocardial Ischemia in aPorcine Model

Yucatan minipigs had toroid shaped ameroid occluders placed on theirproximal left circumflex arteries. The ameroid imbibed water over time,causing constriction of the vessel. Occlusion was verified four weeksafter surgery by contrast enhanced angiography. At that time, eachanimal's chest was reopened, whereupon the region of ischemia wasinjected with a slow release formulation of TP508, i.e.,TP508-containing PGLA microspheres, suspended in a Pluronic gel. ThePLGA microspheres, which were prepared as described in Example 6, gavean initial burst release of drug (50% of load in 24 hours) and thendisplayed controlled release for another 3-4 days, by which time 80% ofthe load had been released. The gel used was 30% w/v Pluronic F68 in0.9% saline. To each milliliter of gel, on ice to reduce the viscosity,3.3 mg of PLGA microspheres were added immediately before injection.This gave a TP508 dose of 100 μg/ml of gel, which was injected into tensites (100 μl per site) in the ischemic area. Controls received PLGAmicrospheres in Pluronic gel without TP508. Baseline, and post-treatmentangiograms and echocardiograms were obtained.

Indices for myocardial wall thickening and cardiac ejection fractionshowed trends that TP508 treated animals tolerated dobutamine-inducedstress better than controls. After three weeks, the animals wereevaluated with contrast enhanced echocardiography. Initial results onthis limited number of animals demonstrated that TP508 treated animalsunder dobutamine stress had a slightly larger increase in ejectionfraction and better maintained wall thickening compared to controls.Thus, this treatment appears to help restore functionality to theischemic heart muscle.

Example 4 TP508 Stimulates Myocardial Revascularization in a RabbitModel

TP508, formulated in sustained release PLGA microspheres, was injectedinto ischemic rabbit myocardium. An ameroid occluder was placed over thelateral division of the left main coronary artery of two rabbits justinferior to the A-V groove, as described in Operschall et al., J. Appl.Physiol. 88:1438 (2000). Two weeks after placement, the animals' chestswere reopened. In one animal, TP508 microspheres in pluronic gel (asdescribed in Example 3) were injected into eight discrete locationswithin, and around, the area served by the occluded vessel. The otheranimal served as an untreated control. Approximately four weekspost-injection, the animals were sacrificed and their hearts fixed in10% buffered formalin for 24 hours. Hearts were then sectioned acrossthe area of interest and stained by hematoxylin-eosin and immunolabelledagainst Von Willebrand Factor (vWF), an endothelial cell marker.

Histology demonstrated that the control animal had significant fibrosisin the area served by the occluder. The TP508 treated heart, on theother hand, had healthy appearing myocardium with a larger number offunctional capillaries with obvious red blood cells.

Example 5 TP508 Suppresses Restenosis in a Hypercholesterolemic RabbitModel

This procedure was designed to provide a system for testing the efficacyof a Test Sample to inhibit neointimal formation and vascular occlusionfollowing angioplasty in hypercholesterolemic New Zealand White Rabbits.The animals were fed a high fat diet consisting of 0.5% cholesterol and2.0% peanut oil for 3 weeks. The animals were pretreated 24 hours priorto surgery; the iliac artery was injured with balloon angioplasty asdescribed; and the animals were are treated with TP-508 for 7 days. Theanimals were maintained on a high fat diet for 4 weeks. Angiography wasconducted prior to balloon angioplasty and at termination of theexperiment. The injured and uninjured iliac arteries were harvested andprepared for histology. Morphometric measurements were made of thelumen, the neointima (if present), and the tunica media.

Test samples of TP-508 were dissolved/diluted in a sterile, pyrogen-freesaline to the desired concentration and administered by intravenousinjection in a 0.2 ml volume one day prior to surgery, the day ofsurgery, and for 6 successive days post surgery.

A 5 cm midline neck incision was made and the right carotid was exposed,proximately ligated, and incised. A 4 Fr Berman Balloon AngiographicCatheter was then introduced into the aorta. A 5 Fr sheath wasintroduced into the aorta via the 4 Fr Berman Balloon AngiographicCatheter. Three ml of blood was collected for cholesterol count. Therabbit was then injected with heparin and more anesthetics (ifnecessary). To visualize the iliac arteries, 6 ml of Hypaque 76% mixedwith 4 ml sterile saline was injected through the catheter. Imaging wasacquired of the iliac arteries (image is marked with grid and scissorsare placed on the right side). The 4 Fr Berman Balloon AngiographicCatheter was removed from the sheath. A 0.014″/3.0 mm×20.0 mm/120 cmBalloon Catheter was then inserted through the sheath into the aorta andto the iliac artery. The balloon was inflated 3 times at 10 ATM for 30seconds with 1 minute intervals. The catheter and sheath were thenremoved. The right carotid artery was ligated with 3.0 silk sutures. Theneck incision was closed with PDS and the skin stapled and dressed withdouble antibiotic ointment.

The test sample(s) or control sample(s) were then administered to therabbit. The Test Sample was diluted in the following manner: 0.3 ml ofsaline was drawn into a 1.0 ml syringe with a 23 G 1″ needle. The volumewas injected into the TP-508 vile. After the TP-508 dissolved, 0.25 mlof the solution was removed and administered. The cannulation tube wasthen flushed with saline. If the rabbit was a control, 0.2 ml of salinewas injected and flushed with additional saline. The rabbit alsoreceived 0.3 ml of Buprenorphine via subcutaneous injection.

After surgery, the rabbit was allowed time to become alert while restingon the heating pad. The rabbit was then returned to his cage and allowedfood and water ad libitum. The rabbits were maintained on the diet for 4additional weeks until sacrifice.

Four weeks post-procedure, both iliac arteries were fixed in situ,harvested and prepared for histology. Digital images were then capturedof the serial histological sections spaced approximately one millimeterapart and morphometric measurements were made of the lumen, theneointima (if present) and the tunica media throughout the region ofinjury.

Histology Summary

Morphohistological analysis of 19 samples were completed using Image-ProPlus and Excel software. Of the 19 samples, 2 demonstrated compromise ofthe external elastic lamina. One sample of the 19 appeared to requireadditional sectioning. Therefore, 16 samples were compared comprising 7treated and 9 saline controls.

The thickness of the restenotic lesion was determined by measuring thearea of the neointima via digital analysis. The tunica media of thevessels was measured similarly. These values were then normalized bysumming the area of these two regions and dividing that result by thearea of a normal (un-injured) media found within the same histologicalslide series. It was verified that there was no significant differencebetween groups in the areas found for the uninjured media.

When comparing treated animals against controls, the extent ofrestenosis was analyzed via three distinct methods: the “single worstvalue” method, the “average lesion thickness” method, and the “averageof all sections” method. The “single worst value” method compares themaximum restenosis value obtained between operated vessels. The “averagelesion thickness” method compares the averages all abnormal pointswithin a well-defined region of injury between operated vessels. Lastly,the “average of all sections” method compares the average thickness ofall samples measured, regardless of whether or not they appeared to bepart of the lesion. The means of these results were tested forstatistical significance via the Student's T-test.

Data Summary

All data analysis was completed using the two-tailed t-test assumingunequal variances. Alpha is 0.05 and the mean difference is assumed tobe 0. Each analysis includes n=7 for treated and n=9 for saline control.The results are summarized in the following Table 1. The “difference”value shown relates to the percentage change of the treated as comparedto the corresponding control. Values noted with an asterix werestatistically significant. TABLE 1 Technique: Single Worst Value AverageLesion Thickness Average of All Sections Measured Area: Treated ControlsDiff. Treated Controls Diff. Treated Controls Diff. Neointima .202 .332−39%* .158 .245 −36% .117 .185 −37%* Media .113 .133 −15%  .123 .152−19% .116 .140 −17%  Neo + Media/Uninjured Media 4.56 7.73 −41%* 4.185.87  −29%* 3.49 5.55 −37%*Conclusion

The data shows that TP-508 significantly suppressed restenosis andvascular occlusion in the hypercholesterolemic rabbit model. This resultis robust in that it is independent of the technique chosen forquantifying the results.

Example 6 Preparation of Polylactic Acid/Polyglycolic Acid CopolymerMicrospheres of TP508

A double emulsion technique was used to prepare microspheres ofpolylactic acid/polyglycolic acid copolymer (PLA/PGA) containing TP508.Briefly, the matrix components were dissolved in methylene chloride andTP508 was dissolved in water. The two were gradually mixed togetherwhile vortexing to form a water-in-oil (W/O) emulsion. Polyvinyl alcohol(0.3% in water) was added to the emulsion with further vortexing to formthe second emulsion (O/W), thereby forming a double emulsion: an O/Wemulsion comprised of PLA/PGA droplets, and within those droplets, asecond disperse phase consisting of TP508 in water. Upon phaseseparation, the PLA/PGA droplets formed discrete microspheres containingcavities holding TP508. To cause phase separation of the microspheres, a2% isopropyl alcohol solution was added. The particles were collected bycentrifugation, and then lyophilized to remove residual moisture. Thecomposition of the matrix was varied to form microspheres with differentrelease kinetics (Table 2). TABLE 2 Composition of different microsphereformulations Polymer % % polyethylene Formulation PLA:PGA M. Wt. TP508glycol A 50:50 46,700 5 0 B 50:50 7,200 5 0 C 50:50 46,700 5 5 D 50:5046,700 5 0 E 75:25 120,000 5 0

The mean diameter of the microspheres was measured in a Coulter counterand the drug entrapment efficiency was measured by spectrophotometricassay at 276 nm following dissolution of a weighed sample ofmicrospheres in methylene chloride and extraction of the released druginto water (Table 3). TABLE 3 Formulation diameter and drug entrapmentefficiency Formulation Diameter, μm TP508 Entrapment, % A 26.0 53.8 B16.2 27.1 C 17.6 58.9 D 23.9 42.6 E 25.8 36.2

To measure TP508 release from the different PLA/PGA matrices, 20 mg ofmicrospheres were placed in 1.0 ml of PBS contained in 1.5 mlpolypropylene microcentrifuge tubes. Tubes were incubated at 37° C. andshaken at 60 rpm. At various times, the tubes were centrifuged and thesupernatant containing released TP508 was removed and frozen forsubsequent analysis. Fresh PBS was added to the microspheres andincubation was continued. TP508 in the supernatant was measured byabsorbance at 276 nm. For each formulation, quadruplicate releasedeterminations were performed. Formulations B and D showed no detectabledrug release during 28 days of incubation at 37° C. The remainingformulations all released detectable amounts of TP508, although in allcases the amount of drug released fell below detectable limits (<1 μg/mgmatrix/day) within 3-4 days. Formulations A and C showed the greatestrelease of TP508, releasing 60-80% of the entrapped drug over 3-4 days.The formulation with the fastest release kinetics, C, was chosen forfurther testing in in vivo studies described in Example 3 and Example 4.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1. A method for promoting cardiac tissue repair in a patient, saidmethod comprising administering to the patient a therapeuticallyeffective amount of an angiogenic thrombin derivative peptide or aphysiologically functional equivalent thereof.
 2. A method ofstimulating revascularization in a patient, said method comprisingadministering to the patient a therapeutically effective amount of anangiogenic thrombin derivative peptide or a physiologically functionalequivalent thereof.
 3. A method of stimulating vascular endothelial cellproliferation in a patient in need of such treatment, said methodcomprising administering to the patient a therapeutically effectiveamount of an angiogenic thrombin derivative peptide or a physiologicallyfunctional equivalent thereof.
 4. A method of inhibiting restenosis in apatient following balloon angioplasty, said method comprisingadministering to the patient a therapeutically effective amount of anangiogenic thrombin derivative peptide or a physiologically functionalequivalent thereof.
 5. A stent coated with an angiogenic thrombinderivative peptide or a physiologically functional equivalent thereof.6. A method of inhibiting vascular occlusion in a patient, said methodcomprising administering to the patient a therapeutically effectiveamount of a thrombin derivative peptide or a physiologically functionalequivalent thereof.